Article

Free radical scavenging activity in

in vitro-derived tissues of Eruca sativa

Toxicology and Industrial Health

2016, Vol. 32(1) 98105
The Author(s) 2013
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DOI: 10.1177/0748233713498450
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Bilal Haider Abbasi1, Jawad Ali1, Mohammad Ali1,

Muhammad Zia1, Saleem A Bokhari2
and Mubarak Ali Khan1
Abstract
Feasible regeneration protocol for economically important plant Eruca sativa was established and 1, 1-diphenyl2-picrylhydrazyl scavenging activity of regenerated tissues was evaluated and compared with plant material collected from the wild. Leaf portions inoculated onto Murashige and Skoog (MS) medium responded to all plant
growth regulators exploited. Optimum callus production was achieved on a combination of 2.0 mg l1 6benzyladenine (BA) 1.0 mg l1 a-naphthalene acetic acid (NAA) and the lowest response was recorded for
0.5 mg l1 gibberellic acid (GA3) 1.0 mg l1 NAA. The callus was subcultured on similar composition/concentrations of plant growth regulators after 4 weeks of culture time. A 5.0 mg l1 6-BA 1.0 mg l1 NAA
produced optimum percentage shoot organogenesis after 4 weeks of subculturing. However, optimum number of shoots per explant was recorded for moderate concentrations (1.0 and 2.0 mg l1) of kinetin. Incorporation of NAA into MS medium-containing GA3 also produced a feasible number of shoots/explant. Similar mean
shoot length was recorded for 2.0 mg l1 kinetin 1.0 mg l1 NAA and optimum concentrations (2.0, 5.0, and
10.0 mg l1) of GA3 1.0 mg l1 NAA. In vitro generated shoots were shifted to MS medium augmented with
indole acetic acid (IAA) for rooting after 4 weeks of subculturing. Moderate concentrations (5.0 mg l1) of IAA
produced feasible rooting. Investigation of radical scavenging activity showed that callus possesses higher levels
of radical scavengers than other plant tissues tested. Phenolics and glucosides are reported to be active components of Eruca sativa phytochemistry.
Keywords
Eruca, organogenesis, cytokinin, auxin, antioxidant, Brassicaceae

Introduction
Eruca sativa commonly known as Rocket belongs to
the family Brassicaceae and is geographically confined
to the Mediterranean region (Chopra et al., 1996; Yaniv
et al., 1998). Rocket was at one time used medicinally,
although it is now used commonly as a salad herb (Lamy
et al., 2008). The plant is reported to have antiscorbutic,
diuretic, stimulant, stomachich, and ribefacient activities (Songsak and Lockwood, 2002). Alqasoumi et al.
(2009) recently found anti-ulcer activity and antiinflammatory effects of Eruca extracts (Yehuda et al.,
2009). Oil extracted from its seed is reported to have
aphrodisiac properties. An economically important
compound, 4-methylthiobutyl glucosinolates have been
extracted from the seeds of E. sativa (Iori et al., 1999;
Zhang et al., 2005). A major compound extracted from
Eruca is erucic acid that is commonly used as lubricant

(Kumar and Tsunoda, 1980). Resistance of plants to

drought, mustard aphid, and other pathogens is also
reported (Tewari et al., 1995). Recently, E. sativa has
shown antioxidant potential and exert protective effect
against mercuric chloride (HgCl2)-induced injuries
(Alam et al., 2007; Alqasoumi, 2010; Barbieri et al.,
2011; Barillari et al., 2005; Sacan et al., 2008).

1
Department of Biotechnology, Faculty of Biological Sciences,
Quaid-i-Azam University, Islamabad, Pakistan
2
Department of Biosciences, COMSATS Institute of Information
Technology, Islamabad, Pakistan

Corresponding author:
Bilal Haider Abbasi, Department of Biotechnology, Faculty of
Biological Sciences, Quaid-i-Azam University, Islamabad 45320,
Pakistan.
Email: bhabbasi@qau.edu.pk

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Abbasi et al.

99

Phytochemistry of Eruca has shown a wide range of

health-promoting chemicals like carotenoids, glucoerucin, vitamin C, fibers, phenolics, and so on (Alqasoumi, 2010; Barillari et al., 2005; Lamy et al., 2008;
Ozdener and Aydin, 2010).
Due to these properties, Eruca is considered as
capable resource for the production of lowmolecular weight antioxidants. Unfortunately, direct
utilization of E. sativa in a breeding program has been
limited owing to the occurrence of strong inappropriateness barriers with other Brassica species. Plant tissue culture provides platform to improve quality and
establish higher frequency regeneration protocol for
this economically important plant species. Unfortunately, few reports are available that were confined
to regeneration from somatic embryos (Zhang et al.,
2005), cotyledonary explants, and mesophyll protoplasts (Surya-Parkash et al., 1989; Sikdar et al.,
1987). However, no report on efficient shoot regeneration from leaf explants in Eruca is available.
The main purpose of this current study was to
establish a feasible regeneration protocol and to evaluate 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity of regenerated tissues and a
comparison with field grown wild plantlets.

Materials and methods

Seeds were provided by Prof. Dr Mir Ajab Khan, Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan. Seeds were decontaminated by the
method developed and reported earlier by Abbasi et al.
(2010, 2011) and Ahmad et al. (2010). Briefly, these
seeds were submerged in 80% ethanol for about 5 min,
0.6% mercuric chloride for about 45s, and washed thoroughly with sterile double distilled water. The decontaminated seeds were placed onto Murashige and Skoog
(MS) medium (Murashige and Skoog, 1962) without any
plant growth regulators (PGR) for germination. Leaf
explants were cut from germinated plantlets after 5
weeks. These leaf explants were sterilized by seed sterilization protocol to prevent endogenous contamination.
For callus formation and shoot organogenesis, leaf
explants (1.01.5 cm2) after sterilization were cultured
on MS medium augmented with either 6-benzyladenine (BA; 0.5, 1.0, 2.0, 5.0, and 10.0 mg l1), kinetin
(Kn; 0.5, 1.0, 2.0, 5.0, and 10.0 mg l1), and gibberellic acid (GA3; 0.5, 1.0, 2.0, 5.0, 10.0 mg l1) either
alone or in combination with 1.0 mg l1 a-naphthalene acetic acid (NAA). Basal MS medium (MS0)
was used as control.

Almost six leaf explants were inoculated on to

*35 ml of coagulated MS medium in 100 ml Pyrex
glass flask. Subsequently, after 28 days of inoculation,
average number of explants response (callus induction)
was recorded. Best growing callus with yellowish green
color was invigorated to MS medium containing similar
concentrations/composition of PGRs for further growth
and development. Data regarding shooting (%), number
of shoots per explant, and mean shoot length were documented after 28 days of subculture callus. Extended
shoots with maximum height and with suitable stem
were carefully shifted to fresh medium having selected
concentrations (1.0, 2.5, 5.0, and 8.0 mg l1) of indole
acetic acid (IAA). Growth room conditions were maintained at 25 + 2 C and 40 + 5 mmol m1 s1 light
intensity with 16 h of photoperiod. Well-rooted plantlets
after 5 weeks of culture were carefully separated from
medium, thoroughly washed with sterile and finally
shifted to pots containing soil for further development.
In vitro regenerated tissues like callus, in vitro
shoots, plantlets, and field-derived plantlets were
exposed to constant radicals of DPPH for activity.
DPPH scavenging efficiency was calculated according
to the modified method of Abbasi et al. (2010). Briefly,
each dried plant tissues, approximately 10.0 mg was
dissolved in 4 ml of methanol and then subsequently
mixed with DPPH (1 mM, 0.5 ml) solution. These mixtures of solution were well vortexed for 15s and then
incubate for 30 min at room temperature. To conclude
final results, the absorbance of samples was checked at
517 nm through spectrophotometer. For background
correction, DPPH solution prepared in methanol had
decayed and hence no longer exhibited purple color
(i.e. 2 mg of butylated hydroxyanisole dissolved in
4 ml of methanol with 0.5 ml of DPPH solution added)
(Amarowicz et al., 2004). The scavenging power of
extracts was determined as percentage of DPPH discoloration using the following well-known equation
Free radical (DPPH) scavenging potential %
100  1AS =AB
where AS indicate the final absorbance of sample
(extract DPPH), when suitable amount of tissue
extract has been supplemented at a particular level;
AB is the absorbance of methanol and DPPH radicals
without the addition of extract.
For statistical exploration, six cultured expants were
kept in each treatment and data were documented from
triplicates. Duncans multiple range test and analysis of
variance were used for assessment between treatment

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Toxicology and Industrial Health 32(1)

Figure 1. Plant regeneration in Eruca sativa (a) callus, (b) shoot regeneration, (c) shoot elongation, and (d) regenerated
plantlets.

90
a

80

% Callus induction response

70

ab ab

ab

b
bc

60
bc

bc

bc

50

bc
c

c
cd

cd

40

cd

cd
d

30

cd

cd

d
de

e
20

e
ef

ef
10

f
0.5Kn
1.0Kn
2.0Kn
5.0Kn
10.0Kn
0.5Kn+NAA
1.0Kn+NAA
2.0Kn+NAA
5.0Kn+NAA
10.0Kn+NAA
0.5GA
1.0GA
2.0GA
5.0GA
10.0GA
0.5GA+NAA
1.0GA+NAA
2.0GA+NAA
5.0GA+NAA
10.0GA+NAA

MS0

0.5 BA
1.0 BA
2.0 BA
5.0 BA
10.0 BA
NAA
0.5BA+NAA
1.0BA+NAA
2.0BA+NAA
5.0BA+NAA
10.0BA+NAA

Plant growth regulators (mg/l)

Figure 2. Effects of various concentrations of BA, Kn, and GA3 with or without 1 mg l1 NAA on percentage callus induction of Eruca sativa. Data were collected after 4 weeks of culture. Values are means of three replicates. BA: 6benzyladenine; Kn: kinetin; GA3: gibberellic acid; NAA: a-naphthalene acetic acid.

means. SPSS for windows was exploited for statistical

calculations.

Results and discussion

Response of leaf explant of rocket
A viable protocol for shoot regeneration from leaf
explants of E. sativa was established (Figure 1). Leaf
explants responded to all of PGRs used (Figure 2).

Significantly higher explant response was recorded for

2.0 mg l1 BA 1.0 mg l1 NAA. In a previous report,
2.0 mg l1 BA 0.5 mg l1 NAA and 2.0 mg l1
Kn 0.2 mg l1 NAA produced 93.3% callus from
cotyledonary explants of E. sativa (Surya-Parkash
et al., 1989). Similar percentage callus induction was
recorded for 5.0 mg l1 BA, 5.0 and 10.0 mg l1 Kn and
combination of 10.0 mg l1 Kn 1.0 mg l1 NAA (Figure 1). Sikdar et al. (1987) found 5.0 mg l1 of

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Abbasi et al.

101

100
a

90
a
80

ab

bc

60

bc

cd

bc

bc

bc

50
40

ab

70
% Shoot organogenesis

ab

cd

bc
cd

cd

cd
cd

cd

cd

de

30

e
20
f f

f f f

0.5Kn+NAA
1.0Kn+NAA
2.0Kn+NAA
5.0Kn+NAA
10.0Kn+NAA
0.5GA
1.0GA
2.0GA
5.0GA
10.0GA
0.5GA+NAA
1.0GA+NAA
2.0GA+NAA
5.0GA+NAA
10.0GA+NAA

MS0

0.5 BA
1.0 BA
2.0 BA
5.0 BA
10.0 BA
NAA
0.5BA+NAA
1.0BA+NAA
2.0BA+NAA
5.0BA+NAA
10.0BA+NAA
0.5Kn
1.0Kn
2.0Kn
5.0Kn
10.0Kn

10

Plant growth regulators (mg/l)

Figure 3. Effects of various concentrations of BA, Kn, and GA3 with or without 1 mg l1 NAA on percentage shoot organogenesis in Eruca sativa. Data were collected after 4 weeks of subculture to MS media with similar composition of plant
growth regulators. Values are means of three replicates. BA: 6-benzyladenine; Kn: kinetin; GA3: gibberellic acid; NAA:
a-naphthalene acetic acid; MS: Murashige and Skoog.

BA 0.75 mg l1 NAA produced optimum morphogenic response in mesophyll protoplasts of E. sativa.
However, an addition of 2, 4 dichlorophenoxy acetic
acid induced embryogenesis in E. sativa (Sikdar et al.,
1987; Zhang et al., 2005). The callus was observed initially green in color which became greenish-yellow with
the passage of time signifying different metabolic
changes in growth. Slater et al., (2011) obtained better
percentage of green callus and shoot production from
hypocotyl segments than cotyledonary explant in
E. sativa. Nonetheless, contrary observations were
made by Batra and Dhingra (1991) and Parkash et al.
(1989). Thus, incorporation of NAA into medium
already having BA enhanced explant response. However, addition of NAA into medium containing Kn/
GA3 did not produce any significant change in results.

Shoot organogenesis
Callus cultures were refreshed to MS medium with similar composition and concentration of PGRs after 4
weeks of culture time. Subsequently, data for shoot

organogenesis were recorded after 4 weeks of this subculturing procedure. The best shoot induction response
(%) was recorded for (2.0 and 5.0 mg l1) of BA
1.0 mg l1 NAA and (5.0 and 10.0 mg l1) of Kn
1.0 mg l1 NAA (Figure 3). However, combination of
2.0 mg l1 BA 0.5 mg l1 NAA produced optimum
percentage shoot induction from cotyledon. Furthermore, GA3 induced shoot organogenesis response at significantly lower levels. Notwithstanding, incorporation
of 1.0 mg l1 NAA into medium containing GA3
enhanced shoot organogenesis frequency. Positive
effects of the addition of 1.0 mg l1 NAA was observed
for percentage shoot organogenesis in E. sativa. These
observations are in agreement with Abbasi et al.
(2010) and contrary to Abbasi et al. (2011) and Ahmad
et al. (2010).
Data on number of shoots/explant indicate that
optimum number of shoots was obtained for moderate
concentrations (1.0 and 2.0 mg l1) of Kn. However,
significant number of shoots/explant was obtained on
moderate concentrations (2.0 and 5.0 mg l1) of BA and
2.0 mg l1 GA3 1.0 mg l1 NAA (Figure 4).

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Toxicology and Industrial Health 32(1)

10
a

Number of shoots/explant

ab

6
cd

bc

bc

cd

cd

c
cd

c
cd
d
de

4
e
3

ef

e
e

de

ef

ef

1
0.5Kn+NAA
1.0Kn+NAA
2.0Kn+NAA
5.0Kn+NAA
10.0Kn+NAA
0.5GA
1.0GA
2.0GA
5.0GA
10.0GA
0.5GA+NAA
1.0GA+NAA
2.0GA+NAA
5.0GA+NAA
10.0GA+NAA

0.5BA+NAA
1.0BA+NAA
2.0BA+NAA
5.0BA+NAA
10.0BA+NAA
0.5Kn
1.0Kn
2.0Kn
5.0Kn
10.0Kn

MS0

0.5 BA
1.0 BA
2.0 BA
5.0 BA
10.0 BA

Plant growth regulators (mg/l)

Figure 4. Effects of various concentrations of BA, Kn, and GA3 with or without 1 mg l1 NAA on number of shoots per
explant in Eruca sativa. Data were collected after 4 weeks of subculture to MS media with similar composition of plant
growth regulators. Values are means of three replicates. BA: 6-benzyladenine; Kn: kinetin; GA3: gibberellic acid; NAA:
a-naphthalene acetic acid; MS: Murashige and Skoog.

Conversely, addition of 1.0 mg l1 NAA into medium

containing Kn inhibited number of shoots/explant significantly but opposite effects were observed for
medium containing GA3. Similar findings for Brassica
rapa were reported earlier (Abbasi et al., 2011). On the
other hand, synergistic effects of auxin with other PGRs
promoted shoot regeneration in Silybum (Abbasi et al.,
2010). Ahmad et al. (2010) produced best shoot induction response on 2.0 mg l1BA 1 mg l1GA3; however, addition of NAA into medium containing BA
inhibited shoot induction response in their study on
Piper spp. Optimum mean shoot length recorded for different concentrations (2.0, 5.0, and 10.0 mg l1) of
GA3 1.0 mg l1 NAA and moderate concentration
(2.0 mg l1) of Kn 1.0 mg l1 NAA (Figure 5). Contrarily, addition of 1.0 mg l1 NAA into medium
containing BA inhibited mean shoot length in the present study (Figure 5). GA3 is also reported to produce
optimum shoot length in Brassica spp (Abbasi et al.,
2011). However, combination of BA and GA3 promoted
shoot length in Piper nigrum (Ahmad et al., 2010). The
concentration of cytokinin was found critical in shoot

induction response in some species (Abdellatef and

Khalafalla, 2007). Furthermore, higher concentration
of the cytokinin inhibited shoot regeneration in tomato
(Ishag et al., 2009). Ahuja et al. (2002) reported positive
synergistic effects of combination of auxin and cytokinin on shoot regeneration in Atropa acuminata. For
E. sativa, however, no data are available to date for its
comparison with present study.

Root organogenesis
Selected shoot after maximum height were carefully
shifted to MS medium supplemented with different
concentration of rooting hormones (1.0, 2.5, 5.0, and
8.0 mg l1) of IAA after 4 weeks of subculture. Optimum rooting response was recorded at moderate
(5.0 mg l1) concentrations of IAA. This conforms
to induce optimum shoot regeneration response in
E. sativa hypocotyl explants (Slater et al., 2011). Previously, it was reported that moderate number of
plantlets were recovered from mature somatic
embryos after transferred to half of the MS medium

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103

7
a
aa

ab

ab
ab

ab
b

bc

c
cd
cd

d
de
de
de

2
de
e

0.5 BA
1.0 BA
2.0 BA
5.0 BA
10.0 BA
NAA
0.5BA+NAA
1.0BA+NAA
2.0BA+NAA
5.0BA+NAA
10.0BA+NAA
0.5Kn
1.0Kn
2.0Kn
5.0Kn
10.0Kn

f
MS0

de

de

0.5Kn+NAA
1.0Kn+NAA
2.0Kn+NAA
5.0Kn+NAA
10.0Kn+NAA
0.5GA
1.0GA
2.0GA
5.0GA
10.0GA
0.5GA+NAA
1.0GA+NAA
2.0GA+NAA
5.0GA+NAA
10.0GA+NAA

Mean shoot length (cm)

Plant growth regulators (mg/l)

Figure 5. Effects of various concentrations of BA, Kn, and GA3 with or without 1 mg l1 NAA on mean shoot length in
Eruca sativa. Data were collected after 4 weeks of sub-culture to MS media with similar composition of plant growth regulators. Values are means of three replicates. BA: 6-benzyladenine; Kn: kinetin; GA3: gibberellic acid; NAA: a-naphthalene
acetic acid; MS: Murashige and Skoog.

% Radical scavenging activity

70
a
60
50

bc

bc

40

30
20
10

incorporated with indole butyric acid (IBA). Induction of rooting on MS basal medium in E. sativa was
also reported previously (Sikdar et al., 1987). NAA
induced adventitious roots in different genotypes of
E. sativa from cotyledonary explants. Gajbhiye
et al. (2011) observed positive effects of reduced levels of sucrose on rooting in Polianthes tuberosa.
Auxin is inevitable component of rooting medium in
most of plant species (Ishaq et al., 2009; Abbasi
et al., 2010, 2011). Our results confirm these findings.

llu
s

at
ed

Ca

ts
pl
an
tle

pl
an
t
W
ild

er
ge
n
Re

Se

ed

de

riv

ed
pl
an

ts

Figure 6. Percent radical scavenging activity in callus,

regenerated plantlets, seed derived plantlets, and wild
plantlets determined by DPPH method. Values are means
of three replicates + SD. DPPH: 1, 1-diphenyl-2-picryl
hydrazyl.

Antioxidant activity
Our results show higher levels of radical scavenging
activity in callus cultures than that in shoots and wild
grown plantlets (Figure 6). Barillari et al. (2005)
concluded involvement of purified glucoreucin in antioxidant activity of E. sativa (Kim and Ishii, 2006). Jin
et al. (2009) correlated consumption of green vegetables
with reduced severity of several health diseases including cardiovascular diseases and cancer. These beneficial

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Toxicology and Industrial Health 32(1)

effects are attributed to a range of phytochemicals

including flavonoids and glucosinolates in Eruca spp.
However, administration of extracts from E. sativa to
rats elicited a substantial defense against renal toxicity
induced by HgCl2 (Alam et al., 2007). Phenolics and flavonoids were shown to play major role in detoxification
of free radicals in aqueous extracts of E. sativa (Jin et al.,
2009; Sacan et al., 2008).
Funding
This research received no specific grant from any funding
agency in the public, commercial, or not-for-profit sectors.

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